ABSTRACT Perturbations in Ca2+ homeostasis, caused by infections, mutations or drugs, can cause severe diseases. Understanding the fundamental Ca2+ signaling pathways that regulate both health and disease are important for advancing medical care and drug development. Rotavirus (RV) is an excellent model system to study how disrupted Ca2+ signaling can result in life-threatening illness. RV is an enteric virus that causes diarrhea and vomiting, which can be fatal in children without supportive care. RV infection is highly localized to the villus tips of the small intestine, and yet this is in contrast to the wide-spread dysregulation of intestinal functions caused during infection. To address this, a major tenant of RV pathogenesis is virus-induced paracrine signaling from infected cells to uninfected cells. RV causes a broad dysregulation in Ca2+ signaling and this is a key mediator of pathogenesis both within the localized niche of RV replication and for the paracrine signaling pathways. Much work has been done to identify the viral protein(s) responsible for the Ca2+ dysregulation, and have identified the RV nonstructural protein 4 (NSP4) as the major effector. NSP4 host Ca2+ signals through two distinct functional domains, one acting inside the infected cell [intracellular NSP4 (iNSP4)] by directly targeting the host cell's ER Ca2+ store, and a second form secreted from infected cells [extracellular NSP4 (eNSP4)] that elicits a Ca2+ signal in uninfected cells. Both iNSP4 and eNSP4 Ca2+ signals are associated with the molecular determinants of diarrhea, namely activation of the enteric nervous system and hyperactivation of Cl- secretion. However, important gaps-in-knowledge remain. First, while NSP4 is the main viral effector of the Ca2+ dysregulation, it does so by exploiting host Ca2+ signaling pathways, but the host proteins and pathways are poorly characterized. Second, while paracrine signaling by eNSP4 (and/or by host-derived molecules induced by RV) is predicted, there has been not directly evidence of an intercellular Ca2+ signal from a RV-infected to an uninfected cell. Our previous and new preliminary work has shown that RV uses iNSP4 to persistently activate the host's the store-operated Ca2+ entry (SOCE) pathway. SOCE is an important Ca2+ signaling pathway in non-excitable cells and a potent activator of Cl- secretion through Ca2+-activated Cl- channels. Aim 1 will investigate how RV exploits SOCE, focusing on microdomains at the cell membrane called ER-PM junctions, where SOCE Ca2+ entry occurs and potential coupling to Cl- secretion. Further, we have generated new cell lines and human intestinal enteroids to established the first experimental systems for direct, Ca2+ imaging-based visualization of RV-induced intercellular ?Ca2+ waves? and identified that this paracrine signal is mediated by purinergic receptors on uninfected cells. In Aim 2, we will investigate the molecular mechanisms for the propagation of the Ca2+ wave and determine whether this purinergic signaling pathway is important for the activation of Cl- and serotonin secretion, and therefore a host mediator of RV diarrhea.